This program focuses on the regulation of nuclear MAP kinase phosphatases. The mitogen-activated protein (MAP) kinases are critical components of the signal transduction pathways that mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses. So far, at least ten MAP kinase family members have been identified in mammalian cells, including the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. ERK1 and ERK2 are the archetypes of the ERK subfamily that are highly activated by growth factors. In contrast, the JNK and p38 subfamilies are preferentially activated by stress including ultraviolet light, heat shock, and lipopolysaccharide, and therefore, are also referred to as stress-activated protein kinases. Once activated, MAP kinases can translocate from the cytoplasm to the nucleus, leading to the phosphorylation of a multitude of transcription factors and altered gene expression. The physiological functions of various MAP kinase subfamilies have been extensively studied in a large number of systems. In general, ERK activation is closely associated with cell proliferation, differentiation and enhanced cell survival after cellular stress, although in certain situations, such as recently described for cisplatin treatment, ERK activation is required for the execution of apoptosis. On the other hand, activation of JNK and p38 is usually associated with enhanced apoptosis and production of inflammatory cytokines, although there are notable exceptions in which JNK/p38 activation is necessary for cell proliferation and differentiation. Since MAP kinase pathways play an important role in regulating many critical cellular processes, the precise regulation of these signaling proteins is crucial for the maintenance of cellular homeostasis. The activities of all MAP kinases are regulated via reversible phosphorylation of the conserved threonine and tyrosine residues in their tripeptide signature motifs by specific MAP kinase kinases and protein phosphatases. In mammalian cells, inactivation of MAP kinases is primarily accomplished by a family of dual-specificity MAP kinase phosphatases (MKPs) that can act on both the phosphothreonine and phosphotyrosine residues. So far, nine distinct mammalian MAP kinase phosphatase family members have been characterized. According to their subcellular localization and patterns of transcriptional regulation, these phosphatases can be roughly divided into two groups. The first group includes MKP-3/Pyst-1, Pyst-2, MKP-4, MKP-5, and M3/6, which are predominantly localized in the cytosol and are therefore thought to mainly control the MAP kinase-regulated events that occur in the cytosol. The second group of enzymes includes MKP-1 (CL100/3CH134), MKP-2, PAC-1, and B23, which are primarily localized in the nuclear compartment. Encoded by immediate early genes, these nuclear MAP kinase phosphatases are rapidly induced by many of the same stimuli that also activate MAP kinases. For this reason, it has been suggested that these MAP kinase phosphatases play an important role in the feedback control of MAP kinase signaling in the nucleus. Recently, it has been reported that several cytosolic MAP kinase phosphatases can interact with their substrate MAP kinases and such an interaction substantially increases their catalytic activities. These studies suggest that catalytic activation of MAP kinase phosphatases mediated through substrate-binding may play an important role in determining their substrate preferences. However, not all cytosolic MAP kinase phosphatases undergo catalytic activation upon interaction with their substrates. For example, although MKP-5 interacts with its substrate MAP kinases through a basic motif in its N-terminal domain, this interaction has little effect on its catalytic activity. Despite the fact that the substrate specificities for many nuclear MAP kinase phosphatases have been studied extensively, relatively little is known about their abilities to interact with their substrates. Even less is known about the mechanisms underlying the interactions, or the effects these interactions have on their biochemical and physiological functions. Although the induction of nuclear MAP kinase phosphatases has been documented in a variety of biological systems, the underlying mechanisms that mediate their transcriptional cregulation remain unclear. Our studies over the past year have concentrated on 2 topics. (1) the catalytic activation of MKP-2 by MAP kinases. MKP-2 has been shown to preferentially inactivate ERK and JNK MAP kinase subfamilies. In order to understand the molecular basis for this substrate selectivity, we have examined MKP-2's interaction with, and catalytic activation by, distinct MAP kinase subfamilies. We found that MKP-2's catalytic activity was dramatically enhanced by ERK and JNK but was only minimally affected by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, while JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the N-terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised MKP-2's catalytic activation by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 while MKP-2 mutants incapable of binding to ERK/p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for MKP-2's substrate preference and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function. (2) The role of histone H3 phosphorylation/acetylation in mediating MKP-1 induction in response to stress. We have shown that MKP-1 mRNA was potently induced by arsenite and ultraviolet light, and modestly increased by heat shock and hydrogen peroxide. Interestingly, arsenite also dramatically induces phosphorylation/acetylation of histone H3 at a global level which precedes the induction of MKP-1 mRNA. The transcriptional induction of MKP-1, histone H3 modification, and elevation in MKP-1 mRNA in response to arsenite are all partially prevented by the p38 MAP kinase inhibitor SB203580, suggesting that the p38 pathway is involved in these processes. Finally, chromatin immunoprecipitation (ChIP) assays reveal that arsenite induces phosphorylation/acetylation of histone H3 associated with the MKP-1 gene and enhances binding of RNA polymerase II to MKP-1 chromatin. ChIP assays following exposure to other stress agents reveal varying degrees of histone H3 modification at the MKP-1 chromatin. The differential contribution of p38 and ERK MAP kinases in mediating MKP-1 induction by different stress agents further illustrates the complexity and versatility of stress-induced MKP-1 expression. Our results strongly suggest that chromatin remodeling after stress contributes to the transcriptional induction of MKP-1.